Project Summary ? Project 1 Phenotypic hallmarks of intoxication with seizure-inducing chemical threat agents include: (1) acute hyperexcitation of neural circuits associated with seizures and status epilepticus (SE); and (2) chronic neuroinflammation and neuropathology that manifest subsequent to seizures. Project 1 is using in vitro models essential for elucidating toxicological and therapeutic mechanisms and for discovering intervention strategies that are more effective and safer than current medical countermeasures (atropine, oxime, high-dose benzodiazepines) for treating acute intoxication and protecting against persistent neuropathology. Our focus is on two classes of threat agents: those that block GABAA receptor (GABAAR) function, e.g., tetramethylene- disulfotetramine (TETS) and picrotoxin (PTX, and organophosphates (OP) that inhibit cholinesterases, e.g., diisopropylfluorophosphate (DFP) and paraoxon (PO). Our hypothesis is that in vitro approaches we developed during the first project period can be used to identify, refine and optimize combinatorial therapies that more effectively target TETS- and OP-triggered seizure mechanisms and control subsequent neuroinflammation. This hypothesis will be tested using in vitro and ex vivo rodent models of both sexes. Aim-1 will determine whether midazolam plus a neurosteroid is more potent than either alone for normalizing TETS and PTX- triggered neuronal network hyperexcitability. In Aim 2 we will test whether subtype selective nicotinic cholinergic receptor (nAChR) inhibitors are superior to our standard midazolam neurosteroid combination or synergize with it in mitigating OP-triggered seizure-like activity in adult neuron/glia cocultures. Our reasoning is based on the known cholinergic mechanisms of seizure initiation. Aim 3 will investigate neuroinflammation by evaluating astrogliosis, microglial activation, and neuropathology following exposure to seizure-inducing chemical threat agents and screen for anti-inflammatory compounds that can mitigate these processes. We have developed two cell-based models (astrocyte/microglia and neuron/glia cocultures) from mouse and rat models of both sexes to quantitatively measure differences in neuroinflammation (astrogliosis and microglia activation) and their relationship to neuropathology over acute and prolonged exposures to chemical threat agents. Results obtained from these three aims will be critical for identifying and prioritizing the most promising candidate therapeutics for mitigating seizure-like activity, neuroinflammation and neuropathology to be testedin vivo by Projects 2 and 3.